This grant will support a continuing research program on protein dynamics, using ultraviolet resonance Raman (UVRR) spectroscopy to characterize protein motions in real time. Understanding proteins as dynamic machines as opposed to rigid structures is key to many potential applications in medicinal chemistry and biotechnology. The site- specific and spatially resolved spectroscopic technique of UVRR will be brought to bear on a wide range of protein mechanisms, from subtly complex folding pathways to functional dynamics such as pore closure and allostery. Folding motions will be investigated in small proteins with different types of secondary structures: CspA (all-? sheet), Villin Headpiece Domain 36 (all-? helix) and FSD (a designed miniprotein with a ?-?-? fold). Functional dynamics will be probed using key models of biological relevance;ferritin (pore/loop closure), ADH (catalysis-related motions), calmodulin (ligand-binding motions) and PTP-ase (loop dynamics). These diverse motions, involving a range of substructures, will support a search for common dynamical principles. Static and time-resolved UVRR experiments will be supported by simultaneous fluorescence monitoring, as well as computational molecular dynamics simulations and static nuclear magnetic resonance, circular dichroism, and UV-Vis spectroscopy. PUBLIC HEALTH RELEVANCE: Proteins are dynamic macromolecules which must be understood at a dynamic as well as structural level in order to tackle biologically relevant problems. Types of protein motions include (un)folding, loop, hinge and pore motions and allostery. All are relevant to a wide array of diseases and disorders caused by protein malfunction. The mechanisms of these motions are amenable to characterization by UV Resonance Raman spectroscopy.